JP4478775B2 - Efficient production method of growth control type recombinant adenovirus vector and kit for production thereof - Google Patents

Description

  Rapid and easy creation of a growth-regulated recombinant adenovirus vector that can be specifically propagated in the target tissue by gene therapy and multiple factors that can be used for these studies, and that can introduce the therapeutic gene specifically in the target tissue The present invention relates to a method and a kit for producing the method.

  Currently, cancer is the top cause of death in Japan and developed countries. In particular, it is difficult to completely overcome cancer metastasis with existing surgical therapy, chemotherapy, and radiotherapy, so in recent years research on new treatment methods, especially gene therapy, is expected and its development has been promoted. Yes. Based on this basic research, gene therapy for cancer has been conducted in many clinical trials in the United States, Japan and other developed countries over the past 10 years, and the number of patients has increased year by year. ing. However, there are no reports that patients were completely cured by gene therapy alone. The biggest cause of this is that most of the currently used gene transfer vectors have been genetically modified so that only a therapeutic gene is introduced after virus infection and no virus growth occurs to ensure safety. This is because the non-propagating virus vector is produced. Although non-proliferating virus vectors are safe, they show superior gene transfer efficiency in in vitro experiments with cultured cells. No gene is introduced into. As long as non-proliferative vectors are used, the problem of cancer recurrence from cancer cells that have not been transfected with genes cannot be overcome, and this is the biggest reason why gene therapy for cancer has not achieved the expected effect in clinical practice. It is.

  As an adenoviral vector (hereinafter abbreviated as ADV) that overcomes this, the region with E1B that specifically grows only in p53 function-deficient cancer cells that are highly dysfunctional in cancer is deleted. Mutant adenovirus (ADV) has been reported (see Non-Patent Document 1). Since then, research has been made on growth-control-type viral vectors that grow in such a cancer-specific manner and do not grow in normal cells. For example, there is an attempt to proliferate specifically for prostate cancer by expressing the E1A gene of adenovirus with a prostate cancer-specific PSA promoter (see Non-Patent Document 2).

However, the growth-regulated viral vectors reported to date are all controlled only by a single factor to specifically target cancer, that is, the single factor can be used between cancer cells and normal cells. Due to the difference in expression, it was devised so that the virus grows only in cancer cells in response to the factor. However, since cancer and normal cells cannot be clearly defined by a single factor at all, existing growth-controlled viral vectors are far from specifically targeting cancer. there were. In order to make this possible, it seems necessary to specifically target cancer simultaneously with multiple factors with different personalities, but there has been no report so far. In addition, each of the conventional growth control virus vectors has to be genetically recombined one by one to prepare each growth control virus vector. Since adenovirus is a long DNA virus of 36 kB, recombination of plasmid vectors containing it is limited in the selection of restriction enzymes, so there are many restrictions on gene recombination and it was impossible to carry out efficiently. For this reason, it is technically difficult to quickly create or modify a large amount of growth control type adenovirus vector, and a large number of growth control type virus vectors can be quickly searched for a vector specifically targeting cancer. It was technically impossible to produce and verify this.
Bischoff JR, et al. Science. 1996 Oct 18; 274 (5286): 373-376 Rodruguez, R., Et al, Cancer Res, 57, 2559-2563, 1997

  The present invention has been made in view of the above circumstances, and in order to search for and produce a virus vector that propagates only in a target tissue such as cancer cells by completely specifying the target tissue such as cancer, it is possible to simultaneously produce multiple factors. The present invention newly provides a method for efficiently, rapidly and easily producing a growth control type recombinant adenovirus vector that specifically targets cancer and the like, and a kit for its production.

The inventors of the present invention have made studies in order to solve the above problems and have completed the present invention.
That is, the present invention comprises, in order from the upstream, the E1A region, at least one protein coding region or the entire E1B region of the E1B region, a poly A signal sequence, and a recombinase recognition sequence, and the endogenous promoter of the E1A region; A growth control unit prepared by deleting an endogenous promoter that regulates the expression of a gene encoding a protein in at least one protein coding region of the E1B region, and inserting a restriction enzyme recognition sequence at each of these deletions. A promoter that specifically expresses in the target tissue is introduced into the restriction enzyme recognition sequence of the vector plasmid containing, respectively, to prepare a growth control vector plasmid. Further, the growth control vector plasmid is deleted from the E1 region. Vector plastics with adenovirus genome Incorporated into phagemid, a gist efficient manufacturing method of a proliferation-regulated recombinant adenoviral vectors, characterized in that to produce the proliferation-regulated adenoviral vector plasmid. Here, the tissue in the target tissue also includes cells.

In the above invention, the Rb protein binding sequence in the E1A region may be deleted. The protein coding region of the E1B region may be a 19 KDa protein coding region and / or a 55 KDa protein coding region. A restriction enzyme that is blunt ended in a restriction enzyme recognition sequence that is inserted into a deletion site of the endogenous promoter that regulates the expression of a gene encoding a protein in the endogenous promoter of the E1A region and at least one protein coding region of the E1B region Each site may be included. The recombinase recognition sequence may be a variant sequence of LoxP also Waso.

  Further, the present invention provides the above-mentioned restriction enzyme recognition sequence of a vector plasmid comprising the above growth control type vector plasmid and a therapeutic gene expression unit prepared by inserting a recombinase recognition sequence and a restriction enzyme recognition sequence in order from the upstream, A recombinase is allowed to act on a first strong therapeutic gene expression promoter or a therapeutic gene expression promoter and a first therapeutic gene expression vector plasmid prepared by introducing the therapeutic gene from the upstream in order, to produce a second therapeutic gene expression vector plasmid, Furthermore, the second therapeutic gene expression vector plasmid is incorporated into a vector plasmid having an adenoviral genome prepared by deleting the E1 region, and a growth control type adenoviral vector plasmid incorporating the therapeutic gene is produced. The therapeutic gene Efficient method for manufacturing a proliferation-regulated recombinant adenoviral vectors and gist was.

  The present invention also provides the restriction enzyme recognition sequence of the above-described growth control type adenovirus vector plasmid and the vector plasmid comprising a therapeutic gene expression unit prepared by inserting a recombinase recognition sequence and a restriction enzyme recognition sequence in order from upstream. A growth control type in which a recombinase is allowed to act on a first strong therapeutic gene expression promoter or a therapeutic gene expression promoter and a first therapeutic gene expression vector plasmid prepared by sequentially introducing the therapeutic gene from upstream. The gist of the present invention is a method for producing a growth-regulated recombinant adenovirus vector incorporating a therapeutic gene, characterized by producing an adenovirus vector plasmid.

  In the above invention, the growth control type adenovirus vector plasmid and the first therapeutic gene expression vector plasmid are mixed and allowed to act on recombinase, and then both are transformed, or the growth control type adenovirus vector plasmid and the first The therapeutic gene expression vector plasmid may be cotransfected into cells expressing recombinase. In the present invention, the recombinase-expressing cell may be a cell expressing adenovirus E1 protein and the recombinase expressed.

In the above invention, the recombinase recognition sequence of the vector plasmid containing the therapeutic gene expression unit may be other than the recombinase recognition sequence of the vector plasmid containing the growth control unit. Furthermore, in the above invention, the drug resistance gene of the vector plasmid containing the growth control unit is different from the drug resistance gene of the vector plasmid containing the therapeutic gene expression unit, and the vector plasmid Ori containing the therapeutic gene expression unit is It may be one that can replicate only in competent cells that express the pir gene.

  In addition, the gist of the present invention is a vector plasmid containing a growth control unit used in the above-described method for efficiently producing a growth control type recombinant adenovirus vector. By using a vector plasmid containing this growth control unit, any two or more promoters that are specifically expressed in the target tissue can be introduced into restriction enzyme recognition sequences to easily produce a growth control vector plasmid. A recombinant adenovirus vector can be easily produced.

  The present invention also provides a vector plasmid containing a growth control unit and a vector plasmid having an adenovirus genome lacking the E1A region, which is used in the above efficient method for producing a growth control type recombinant adenovirus vector. The gist of the preparation kit is as follows. With this production kit, it is possible to easily produce a growth-regulated recombinant adenovirus vector that grows only in the target tissue using any two or more promoters that are specifically expressed in the target tissue.

  In addition, the present invention provides a vector plasmid including a growth control unit and a vector plasmid including a therapeutic gene expression unit for use in an efficient production method of a growth control type recombinant adenovirus vector into which the above therapeutic gene is incorporated. And a vector plasmid having an adenovirus genome lacking at least the E1A region. This preparation kit facilitates a growth-regulated recombinant adenovirus vector that has only two or more promoters that are specifically expressed in the target tissue and any therapeutic gene that is expressed in the target tissue, and that only grows in the target tissue Can be made.

According to the efficient production method of the growth control type recombinant adenovirus vector of the present invention, a promoter that is expressed only in the target tissue can be easily inserted into the adenovirus. Thus, it is possible to efficiently and rapidly produce an adenovirus vector that can be propagated specifically only in a target tissue by general multifactors and can suppress the growth of cancer cells and the like.
Further, according to the efficient production method of the growth control type recombinant adenovirus vector into which the therapeutic gene of the present invention is incorporated, a promoter that is expressed only in the target tissue and the therapeutic gene can be easily inserted into the adenovirus. Controls growth outside of the tissue, specifically propagates only in the target tissue by tissue-specific multifactors, suppresses the growth of cancer cells, etc., and selectively introduces therapeutic genes only in the target tissue Can be expressed.
Therefore, the present invention makes it possible to easily create an adenovirus vector that can specifically target only cancer cells by freely and efficiently producing various growth control-type recombinant adenovirus vectors that are specified by multiple factors. It can be created and analyzed, and it can be widely applied to biological research and basic cancer research in general that tackle the fundamental problems of cancer such as the difference between cancer and normal cells.
Moreover, according to the preparation kit of the present invention, production of a growth control type recombinant adenovirus vector into which an arbitrary promoter that is expressed only in the target cell is incorporated, or a growth control type recombinant adeno virus into which an arbitrary therapeutic gene is incorporated. Viral vectors can be easily prepared.

  As the adenovirus used in the present invention, human adenovirus type 5, human adenovirus type 2, other types of human adenoviruses, adenoviruses of other animal species, and the like can also be used.

A vector plasmid containing a growth control unit can be prepared using a general method in genetic engineering as follows.
That is, the E1A region containing a poly A signal sequence that does not contain an endogenous promoter is amplified by PCR using a plasmid containing at least the 5 ′ genome of adenovirus as a template, and the resulting PCR product and cloning vector are used as restriction enzymes. And then ligated and incorporated into a cloning vector to obtain pΔPr.E1A (hereinafter Pr is a promoter). An arbitrary restriction enzyme recognition sequence is added to the PCR sense primer and the anti-sense primer, and an arbitrary restriction enzyme recognition sequence is inserted into the deletion site of the endogenous promoter. The plasmid used as a template is not particularly limited as long as it contains at least the 5 ′ genome of adenovirus. There is no particular limitation on the cloning vector.

  Next, PCR is used to amplify at least one protein coding region of E1B lacking an endogenous promoter that regulates the expression of the gene encoding the protein in the protein coding region using a plasmid containing the adenovirus genome as a template in the same manner as described above. Then, after digesting the PCR product and the above pΔPr.E1A with a restriction enzyme, ligation and incorporation into pΔPr.E1A yields pΔPr.E1A-ΔPr.XkΔpA (hereinafter, pA is a poly A signal sequence). XK is at least one protein coding region encoding several proteins such as X55KDa and 19KDa of the E1B region.

  A plasmid containing a poly A signal sequence is used as a template and amplified by PCR in the same manner as described above. The obtained PCR product and the above pΔPr.E1A-ΔPr.XkΔpA are digested with restriction enzymes and ligated to give pΔPr.E1A- Incorporate into ΔPr.XkΔpA to obtain pΔPr.E1A-ΔPr.XKpA. A recombinase recognition sequence is added to the PCR primer, and the recombinase recognition sequence is inserted downstream of the poly A signal sequence. The plasmid containing the poly A signal sequence is not particularly limited.

  The vector plasmid pΔPr.E1A-ΔPr.XKpA containing the growth control unit thus obtained is an endogenous promoter that regulates the expression of a gene encoding a protein in the E1A region endogenous promoter and the E1B region protein coding region. In order to insert a restriction enzyme recognition sequence at these deletion sites. The restriction enzyme recognition sequence is preferably provided with a restriction enzyme site that digests to blunt ends, such as SnaBI, EcoRV, HaeIII, AluI, and SmaI.

  In addition, it has been reported that mutant adenoviruses lacking the Rb protein binding sequence (923-947 bp, 24 bp) inside E1A perform tumor-specific virus growth (Heise, C. et al, Nat Med. 2000; 6 (10): 1134-9), the Rb protein binding sequence may be deleted in the E1A region. E1AΔ24 lacking the Rb protein binding sequence is a mutation using the PCR method using primers designed from the Rb protein binding sequence using the above E1A region as a template and a plasmid containing the above adenovirus genome. Obtained by the introduction method (Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 8.5.7-8.5.9, 1999). The vector plasmid pΔPr.E1AΔ24- containing E1AΔ24 containing the above growth control unit was digested with a restriction enzyme and then ligated to delete the Rb protein-binding sequence after digesting the vector plasmid pΔPr.E1A-ΔPr.XkpA. ΔPr.XKpA is obtained.

  A recombinase recognition sequence is a base sequence that is recognized by a specific DNA recombination enzyme, recombinase, and DNA recombination reactions such as DNA strand breaks, substitutions, and bonds between the two recombinase recognition sequences. A specific base sequence that produces The recombinase expression gene is a gene that expresses recombinase and recombinase Cre derived from bacteriophage P1 that recognizes other mutant sequences such as the recombinase recognition sequence loxP or LoxH (Sternberg et al. J. Mol. Boil. Vol. 150, 467 -486 (1981)), recombinase FLP (Babineau et al., J. Biol. Chem. Vol. 260, 12313-12319 (1985)) derived from yeast (Saccharomyces cerevisiae) that recognizes the recombinase recognition sequence FRT, Tigo Saccharomyces As a representative example, a gene expressing R (Matsuzaki et al., Mol. Cell. Biol. Vol. 8, 955-962 (1988) derived from Loei's pSR1 plasmid can be mentioned, but not limited thereto.

  Alternatively, pΔPr.E1A-ΔPr.E1BpA may be used in which the E1B region of the vector plasmid containing the growth control unit is the entire region of E1B. A primer designed based on the base sequence of E1B is used to amplify by PCR using a plasmid containing at least the 5 ′ genome of the adenovirus as a template to obtain a PCR product. The vector plasmid pΔPr.E1A containing the PCR product and the growth control unit, in which the vector plasmid pΔPr.E1AΔ24-ΔPr.XKpA containing the growth control unit was digested with restriction enzymes and then ligated to replace the entire E1BXK and E1B sequences. -Obtain ΔPr.E1BpA.

The restriction enzyme recognition sequence inserted at each deletion site of the endogenous promoter of the vector plasmid pΔPr.E1A-ΔPr.XKpA containing the prepared growth control unit is inserted with a tissue-specific promoter. A growth control vector plasmid can be prepared.
The promoter specifically expressed in the target tissue is a CEA (Carcinoembryonic antigen, carcinoembryonic antigen) promoter (Mol. Cell. Biol., 10 () that is specifically expressed only in cancer cells if the target tissue is cancer cells. 6), 2738-2748, 1990), E2F promoter (Neuman, E., et al, MoI. Cell. Biol., 14 (10), 6607-6615, 1994), OC (osteocalcin) promoter (Morrison, NA. , et al, Science, 246, 1158-1161, 1989), FLK-1 promoter specific for malignant melanoma, fibrosarcoma, etc. (Xie, B, et al, Br J Cancer, 81, 1335-1343, 1999) , A VEGF promoter specific for lung cancer (Koshikawa, N, et al, Cancer Res., 60, 2936-2941,2000), a c-Myc promoter specific for small cell lung cancer (Kumagai, T., et al , Cancer Res., 354-358, 1996), SLPI promoter specific for lung cancer, ovarian cancer, etc. (Garver, RI, et al, Gene Ther, 1,46-50, 1994), specific for prostate cancer PSA promoter (Latham, JP, et al, Cancer Res , 60,334-342,2000), Tyrosinase promoter specific for malignant melanoma (Vile, RG, et al, Cancer Res, 53, 962-967, 1993), AP-2 promoter specific for breast cancer (Pandha, HS, et al, J Clin Oncol, 17, 2180-2189, 1999), and TERT promoters specific to many cancers including brain tumors (Takakura M, et al, Cancer Res, 59, 551-557, 1999).

  This growth-regulated vector plasmid is incorporated into a plasmid having an adenovirus genome prepared by deleting the E1 region, and growth in other tissues is controlled by two promoters (multi-factor) that are expressed specifically in tissues. A growth-regulated adenovirus vector plasmid that grows only in tissues can be produced. The produced growth control-type adenovirus vector plasmid is a cell that constantly produces adenovirus E1 region protein, such as 293 cells derived from human fetal kidney cells (Graham, FL, et al, J. Gen. Virol ., 36 (1): 59-74) can be transfected and propagated. A plasmid containing an adenovirus genome prepared by deleting the E1 region is changed to a adenovirus cancer cell by changing the fiber that regulates adenovirus infection to a ligand that is highly expressed in cancer cells. Therefore, the plasmid fiber having the adenovirus genome prepared by deleting the E1 region may be changed to such a ligand.

In addition, a vector plasmid containing a therapeutic gene expression unit can be prepared using a general method in genetic engineering as follows.
A plasmid having a constitutive strong expression promoter or an expression promoter that regulates the expression of a therapeutic gene and a restriction enzyme recognition sequence for incorporating the therapeutic gene is prepared downstream of LoxP or its mutant sequence.
Also, if the drug resistance gene of the vector plasmid containing the therapeutic gene expression unit and the drug resistance gene of the vector plasmid containing the growth control unit are the same, either one is recombined so that both become different drug resistance genes, Efficient and easy selection of only plasmids that have been correctly recombined by recombination by ensuring that the vector plasmid Ori containing the therapeutic gene expression unit can only replicate in competent cells that express pir genes such as R6Kγ It becomes possible.

  The restriction enzyme recognition sequence of the vector plasmid containing the generated therapeutic gene expression unit includes a constant strong expression promoter or a therapeutic gene expression promoter for treating target tissues such as cancer cells and the therapeutic gene in order from the upstream. Insert to create a first therapeutic gene expression vector plasmid. The constant strong expression promoter means a promoter that constantly and strongly expresses most genes including a therapeutic gene. For example, a CA (hybrid promoter of cytomegalovirus enhancer and chicken β actin promoter) promoter or CMV ( Cytomegalovirus early gene enhancer / promoter). A therapeutic gene refers to a gene that treats a disease at the molecular level by being introduced into a diseased tissue and expressed. For example, cancer suppressors such as P53 gene, IL-2, etc. Examples include cytokine genes, suicide genes such as HSV-tk gene, apoptosis-inducing genes such as Fas, and antisense genes. The therapeutic gene expression promoter is a promoter that regulates these therapeutic genes.

  A recombinase is allowed to act on the growth control vector plasmid and the first therapeutic gene expression vector plasmid to recombine the two, so that a promoter that is specifically expressed in the target tissue and a therapeutic gene that is expressed in the target tissue are The introduced second therapeutic gene expression vector plasmid can be prepared. By incorporating this second therapeutic gene expression vector plasmid into a plasmid having an adenoviral genome prepared by deleting the E1 region, a growth control-type adenoviral vector plasmid incorporating the therapeutic gene can be prepared.

  In addition, the above-mentioned growth control type adenovirus vector plasmid in which the above therapeutic gene is not incorporated and the above first therapeutic gene expression vector plasmid are mixed and allowed to act on recombinase. Can be prepared as a growth control type adenovirus vector plasmid. A recombinase is allowed to act on the growth control type adenoviral vector plasmid and the first therapeutic gene expression vector plasmid, and then both are transformed to produce a growth control type adenoviral vector plasmid in which the therapeutic gene is incorporated. good.

  The above-obtained growth-regulated adenovirus vector plasmid into which the therapeutic gene is not integrated or a growth-regulated adenoviral vector plasmid into which the therapeutic gene has been integrated, such as a cell that constantly produces an adenovirus E1 region protein, such as human By transfecting 293 cells derived from fetal kidney cells and the like, a growth-regulated recombinant adenovirus vector can be produced.

  In addition, the growth control type adenovirus vector plasmid in which the above therapeutic gene is not incorporated and the first therapeutic gene expression vector plasmid described above are cotransfected into a cell expressing recombinase, so that the growth control type adenovirus vector plasmid is obtained. May be produced. In this case, if a cell that expresses recombinase and expresses adenovirus E1 region protein, for example, a cell that expresses recombinase in 293 cells or the like, is co-transfected, the growth control type assembly in which the therapeutic gene is directly incorporated. Alternative adenoviral vectors can be made.

  Propagation, collection, and purification of growth-regulated recombinant adenovirus vectors that do not incorporate the therapeutic gene and growth-regulated recombinant adenovirus vectors that incorporate the therapeutic gene are the same as for the E1 region protein of adenovirus. Cells, such as 293 cells derived from human embryonic kidney cells, can be used in general handling of adenoviral vectors.

Next, in order to understand the principle of the efficient production method of the growth control type recombinant adenovirus vector according to the present invention, a schematic diagram will be described based on FIG. 1 with an example of targeting cancer cells. In the figure, (1) is a promoter that regulates the expression of E1A, (2) is a promoter that regulates the expression of E1B, (3) is a deletion of the Rb protein binding domain, (4) is E1B55KDa and / or Deletion of E1B19KDa, (5) shows the promoter regulating the expression of the therapeutic gene, (6) shows the therapeutic gene, and (7) shows the modification of the adenovirus structural protein gene.
(1) Cancer-specific growth control can be performed by freely replacing any promoter specifically expressed in cancer cells in (1) and (2). E1A is the first region to be transcribed after infection with adenovirus, and if this is not transcribed, efficient viral propagation of adenovirus will not occur. Therefore, conventional non-proliferating adenovirus vectors have deleted the gene of this E1A region and introduced the desired therapeutic gene there. On the other hand, the E1B region encodes several proteins such as 55 KDa and 19 KDa, but this region is also transcribed early after the transcription of E1A, and the transcription and activation of the E1B region also contributes to efficient virus propagation of adenovirus. It is said to be important. Therefore, the endogenous promoter of the virus was removed from E1A and E1B, and a restriction enzyme recognition sequence (multicloning site) was inserted into that region so that other promoters could be introduced easily. By simply replacing two different promoters that are highly expressed specifically in cancer cells, adenovirus growth becomes cancer-specific.
Furthermore, by deleting the Rb protein binding domain in (3) or by deleting the E1B55KDa and / or E1B19KDa region in (4), the mechanism has not yet been fully clarified scientifically. There are separate reports that adenovirus has grown specifically for cancer. By preparing the plasmids with and without (3) and (4) independently, the factors (3) and (4) can be easily combined with the factors (1) and (2) to identify cancer. Can be targeted. Thus, cancer-specific growth control is enabled by four completely independent factors.

On the other hand, by using a cancer-specific promoter, a gene showing a significant effect only on cancer, etc. in (5) and (6), respectively, cancer can be specifically targeted from the viewpoint of expression and effect of a therapeutic gene.
In (7), genes encoding adenovirus structural proteins, including fibers in adenovirus plasmid vectors containing the adenovirus genome, can be easily changed.

A feature of the present invention is that three independent plasmid vectors are used, such as a vector plasmid containing a growth control unit, a vector plasmid containing a therapeutic gene expression unit, and a plasmid containing an adenovirus genome lacking the E1A region. Thus, genetic recombination for the production of the desired growth-controlled recombinant adenovirus vector can be performed very efficiently and freely. In other words, these three plasmids were prepared by independently recombination, and they could be easily recombined and inserted into each other, and each factor combination and combination could be freely and quickly performed. It is. In other words, this can be made more possible by the following measures.
(a) By changing the replication origin placed on one plasmid to one that works only with special E. coli, and further utilizing the difference in antibiotic resistance genes, the efficient selectivity of the target correctly recombined plasmid Is possible. (b) Using a recombination reaction, a therapeutic gene can be easily transferred to an adenovirus vector in Escherichia coli during gene recombination or in 293 cells during adenovirus production. (c) A vector plasmid containing a growth control unit or a recombination plasmid containing a growth control unit and a therapeutic gene expression unit can be freely ligated into a plasmid containing an adenovirus genome by using a restriction enzyme site. Insertion is possible. That is, in this way, each of the three types of target plasmids can be uniquely recombined, and a growth control-type recombinant adenovirus vector having an appropriate combination can be easily prepared according to each purpose. In addition, the completed adenovirus vector plasmid can be easily modified. A shuttle vector can also be used as the vector of the present invention.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to a following example.

Example 1 (Preparation of a vector plasmid containing a growth control unit for expressing the E1 region of adenovirus with an arbitrary promoter)
Construction of adenovirus vector As the plasmid vector, pHM5 was used. pHM5 is a cloning vector having restriction enzyme recognition sequences of SphI, PstI, HincII, XbaI, BamHI, KpnI, SacI, EcoRI, and was provided by Dr. Mark A Kay (Stanford University).
Human Gene Therapy, 10: 2013-2017, 1999). Plasmid pXC1 containing the human 5 type adenovirus genome 5 ′ sequence was purchased from Microbix (Tronto, Canada). It includes the coding region from the E1A protein of the adenovirus genome to the polyadenylation signal, the region not containing the endogenous promoter (474 to 1658 bp), and the coding region of the E1B19KDa protein (1684 to 2285 bp) only. The regions that do not contain the promoter and polyadenylation signal are each pXC1 as a template, and the Bovine growth hormone polyadenylation signal sequence (BGHp: bovine growth factor polyadenylation signal sequence) is pRc / RSV.
(Invitrogen, catalog number A-150307) was used as a template, and was amplified by the PCR method using KOD DNA polymerase (Toyobo, catalog number KOD-101) with the primer set shown in FIG. 2 and cloned. The E1A sense primer used here (S-E1A, SEQ ID NO: 1) is a recognition sequence of each restriction enzyme of SphI, NotI, SnaBI, MluI, and the same antisense primer (AS-E1A, SEQ ID NO: 2). The recognition sequences for SalI and AvrII, the E1B19KDa sense primer (S-E1B19K, SEQ ID NO: 3) are the recognition sequences for the AvrII, SalI, NdeI, EcoRV and MfeI restriction enzymes, and the same antisense primer (AS-E1B19K, SEQ ID NO: 4 ) For BamHI recognition sequence, BGHpA sense primer (S-BGHpA, SEQ ID NO: 5) for BamHI recognition sequence, and antisense primer (AS-BGHpA, SEQ ID NO: 6) for 34 bp LoxH sequence and EcoRI Each recognition sequence has been added (see Figure 2 for the sequence of each primer and the conditions for each PCR). Next, the E1A coding region PCR product and pHM5 obtained above were digested with restriction enzymes SphI (Takara Shuzo, catalog number 1180A) and SalI (Toyobo, catalog number SAL-111), and T4 DNA ligase (Takara Shuzo, Ligation was carried out according to catalog number 6022) to produce plasmid pΔPr.E1A in which the coding region of E1A was incorporated into pHM5. In addition, the PCR product containing the coding region of E1B19K obtained above and pΔPr.E1A were digested with SalI and BamHI (Toyobo, catalog number BAH-111), ligated with T4 DNA ligase, and plasmid pΔPr.E1A-ΔPr .19KΔpA was produced. Further, BGHpA and pΔPr.E1A-ΔPr19KΔpA of the PCR product obtained above were digested with BamHI and EcoRI (Toyobo, catalog number ER0271), ligated, and plasmid pΔPr.E1A-ΔPr.19K-BGHpA (hereinafter referred to as BGHpA). Was omitted, and expressed as pΔPr.E1A-ΔPr.19K). The DNA sequence of pΔPr.E1A-ΔPr.19K is correct with a DNA sequencer (Applied Biosystems, ABI PRISM 310 Genetic Analyzer) to eliminate the possibility of base sequence mutations that may occur due to the use of PCR for cloning. It was confirmed that it was a sequence and that the target DNA construct was prepared. Thereafter, DNA cloned using the PCR method was always confirmed using a DNA sequencer in the same manner.

  The plasmid pΔPr.E1A-ΔPr.19K prepared in this way is a plasmid having a background of pHM5, and in the upstream, recognition of restriction enzymes SphI, NotI, SnaBI, and MluI as multicloning sites. Sequence, coding region of E1A protein, recognition sequence of SalI, NdeI, EcoRV and MfeI restriction enzymes, coding region of E1B19KDa protein, BGHpA and LoxH sequences as multiple cloning sites. That is, a promoter can be freely inserted upstream using the multiple cloning site upstream of E1A and E1B19KDa. In addition, the E1A and E1B19KDa upstream multicloning sites use one restriction enzyme site, SnaBI and EcoRV, respectively, so that any promoter sequence excised with any restriction enzyme can be blunted. If ligation is performed after the stump treatment, the fragment can be easily and reliably incorporated into the vector plasmid pΔPr.E1A-ΔPr.19K containing the unit for growth control.

  In order to produce a mutant E1A lacking the Rb protein binding sequence, a mutagenesis method by PCR using pXC1 shown in FIG. 3 as a template (Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 8.5.7- 8.5.9, 1999) was carried out as follows to delete the 923 to 947 bp region of pXC1 corresponding to the Rb protein binding sequence. First, as a primary PCR, a sense primer (S-E1A, SEQ ID NO: 1) in which recognition sequences of SphI, NotI, SnaBI, and MluI restriction enzymes are added to the E1A5 'end sequence used for E1A cloning, and an Rb binding sequence PCR with antisense primer AS-Δ24 (SEQ ID NO: 8) with 10 bp immediately before (913 to 922 bp) and 20 bp immediately after (947 to 966), and 20 bp immediately before Rb binding sequence (903 to 922 bp) and immediately after PCR with 10-bp (947-956) sense primer S-Δ24 (SEQ ID NO: 7) and antisense primer (AS-E1A, SEQ ID NO: 2) with restriction enzyme SalI recognition sequence added to the E1A3 'end And 475 bp and 726 bp PCR products were obtained, respectively. When both PCR products are mixed and used as a template, and secondary PCR is performed with sense primer S-E1A and antisense primer AS-E1A, the combined product, that is, E1A lacking the Rb protein binding sequence (24 bp) (E1AΔ24) was obtained. The primer sequences and PCR reaction conditions are shown in FIG. This E1AΔ24 was digested into pΔPr.E1A-ΔPr.19K with restriction enzymes SphI and SalI (Toyobo, catalog number SAL-111), inserted with DNA ligase (Takara Shuzo, catalog number 6022), and replaced with wild-type E1A. PΔPr.E1AΔ24-ΔPr.19K. Thus, pΔPr.E1AΔ24-ΔPr.19K is a vector comprising a growth control unit having the same base sequence and structure except that the Rb protein binding sequence inside E1A is removed from pΔPr.E1A-ΔPr.19K. It is a plasmid.

  A shuttle vector using not only the 19 KDa protein portion of the E1B gene but also all the wild-type adenovirus E1B sequences including 55 KDa and other regions was recombined as follows. First, a sense primer (S-E1B-2015, SEQ ID NO: 9) having the same sequence as 20 bp (2015 to 2034 bp of pXC1) from 14 bp upstream to 33 bp upstream from the KpnI recognition sequence in E1B, and polyadenyl of wild type E1B PXC1 using an antisense primer (AS-E1B-4073, SEQ ID NO: 10) with LoxH sequence (34 bp) and EcoRI recognition sequence added to the 24 bp sequence from the 3 'end (4050-4073 bp of pXC1) Amplification was carried out by PCR using KOD DNA polymerase (Toyobo, catalog number KOD-101) as a template, and E1B 2015-4073 bp was cloned. The primer sequences and PCR reaction conditions are shown in FIG. This PCR product was digested with restriction enzymes KpnI (Toyobo, catalog number KPN-111) and EcoRI (Toyobo, catalog number ECO-111), and pΔPr.E1A-ΔPr.19K was similarly digested with KpnI and EcoRI. It was inserted by DNA ligase (Takara Shuzo, catalog number 6022). Thus, the entire E1B19K and E1B sequences were exchanged, and the resulting plasmid was designated as pΔPr.E1A-ΔPr.E1B. PΔPr.E1A-ΔPr.E1B produced in this way is designed to be expressed by an external promoter using the entire E1A and E1B proteins, and pΔPr.E1A-ΔPr.19K is a part of E1B19KDa. However, pΔPr.E1A-ΔPr.E1B is a plasmid having the same base sequence and structure except that the coding region of all E1B is used. In the same manner, a vector plasmid pΔPr.E1AΔ24-ΔPr.E1B containing a growth control unit having the entire E1B sequence was obtained from pΔPr.E1AΔ24-ΔPr.19K lacking the Rb protein binding sequence in E1A.

Example 2 (Insertion of an arbitrary promoter into a vector plasmid containing a growth control unit, production of a growth control vector plasmid)
Several experimental examples in which any desired promoter was actually inserted into the vector plasmid containing the growth control unit prepared above are described below.
First, the CMV (Cymomegalovirus) promoter (Boshart, M., et al, Cell, 41,521-530, 1985, Nelson, JA., Et al, Mol. Cell. Biol., 7, 4125-4129, 1987) was used for E1B19K. A plasmid inserted upstream was prepared. The CMV promoter consists of a plasmid pRc / CMV (Invitrogen, catalog number A-150307) as a template, a sense primer with a SalI recognition site (S-CMVp, SEQ ID NO: 11), and an antisense primer with an MfeI recognition site (AS -CMVp, SEQ ID NO: 12), PCR was performed (sequence of each primer and PCR conditions are described in FIG. 5). This PCR product (231 to 893 of pRc / CMV) and plasmids pΔPr.E1A-ΔPr.19K or pΔPr.E1AΔ24-ΔPr.19K were respectively prepared with SalI (Toyobo, catalog number SAL-111) -MfeI (New England Biolabs, catalog number R0589S), ligated with T4 DNA ligase, inserted CMV promoter upstream of E1B19K, and each growth control type of pΔPr.E1A-CMV-19K and pΔPr.E1AΔ24-CMV-19K A vector plasmid was prepared.

Next, the CEA promoter was inserted upstream of E1A of pΔPr.E1A-CMV-19K and pΔPr.E1AΔ24-CMV-19K. First, adenovirus AxCEAprTK with CEA promoter (provided by RIKEN DNA Bank) 10μl (5.6x10 ¹⁰ pfu / μl), 10% SDS (Sodium dodecyl sulfate, Wako Pure Chemicals, catalog number 199-07145) 10μl, 0.5M The mixture was mixed with 8 μl of EDTA (Ethylenediaminetetraacetic acid, Wako Pure Chemicals, catalog number 311-90075) and 170.75 μl of distilled water and stirred well. Further, 1.25 μl of 20% proteinase K (Roche Diagnostics GmbH, catalog number 745725) was added, digested at 56 ° C. for 2 hours, purified with phenol / chloroform and precipitated with ethanol. By this operation, 2 μg of genomic DNA of AxCEAprTK was collected. Sense using the restriction enzyme NotI recognition sequence in the portion from 424 bp upstream to 2 bp upstream from the start codon of the CEA coding region (Osaki, T., et al, Cancer Res, 54, 5258-5261) using this DNA as a template PCR was performed with a primer (S-CEAp, SEQ ID NO: 13) and an antisense primer (AS-CEAp, SEQ ID NO: 14) with a restriction enzyme MluI recognition sequence. 5). This PCR product was digested with MluI (Toyobo, catalog number MLU-101) and NotI (Toyobo, catalog number NOT-111), and similarly pΔPr.E1A-CMV-19K, pΔPr.E1AΔ24- digested with MluI and NotI. Ligation reaction was performed with CMV-19K and T4 DNA ligase to prepare pCEA-E1A-CMV-19K and pCEA-E1AΔ24-CMV-19K. The thus prepared pCEA-E1A-CMV-19K and pCEA-E1AΔ24-CMV-19K are growth control type vector plasmids in which E1A or E1AΔ24 is expressed by the CEA promoter and E1B19K is expressed by the CMV promoter ( (See Figures 6-1 and 6-2).

Next, a construct was prepared in which the E2F promoter was inserted as each of the E1A and E1B19K promoters. The E2F promoter is Dr. Fine, H. (National Cancer Institute,
The plasmid pABS.4: E2F-GFP provided by Bethesda, MD was excised with restriction enzymes SpeI (Toyobo, catalog number SPE-101) and XhoI (Toyobo, catalog number XHO-101), and T4 DNA polymerase (MBI) , Catalog number EP0061). The above-mentioned pΔPr.E1A-CMV-19K and pΔPr.E1AΔ24-CMV-19K are digested with SalI, blunt-ended with T4 DNA polymerase, and calf intestine alkaliphosphatase (CIP: cattle intestine alkaline phosphatase, Takara Shuzo, catalog number) to prevent self-ligation 2250A) and ligated with the excised E2F promoter with T4 DNA ligase to prepare pE2F-E1A-CMV-19K and pE2F-E1AΔ24-CMV-19K. The pE2F-E1A-CMV-19K and pE2F-E1AΔ24-CMV-19K thus prepared are growth control vector plasmids in which E1A or E1AΔ24 is expressed by the E2F promoter and E1B19K is expressed by the CMV promoter ( (See Figures 6-1 and 6-2).

  In the same manner, the CMV promoter part of pCEA-E1A-CMV-19K and pCEA-E1AΔ24-CMV-19K was digested with SalI and MfeI, and the remaining vector part was treated with T4 DNA polymerase and CIP. Ligation was performed with the excised E2F promoter and T4 DNA ligase to prepare plasmids pCEA-E1A-E2F-19K and pCEA-E1AΔ24-E2F-19K, respectively. The thus prepared pCEA-E1A-E2F-19K and pCEA-E1AΔ24-E2F-19K are growth control-type vector plasmids in which E1A or E1AΔ24 is expressed by the CEA promoter and E1B19K is expressed by the E2F promoter ( (See Figures 6-1 and 6-2).

Next, a construct was prepared in which the OC (osteocalcin) promoter was inserted as the E1A promoter. First, human osteosarcoma SaOS-2 (provided by Tohoku University Institute of Aging Medicine, Medical Cell Resource Center) 5x10 ⁶ were collected by trypsinization and collected using Sapagene (Sanko Junyaku, catalog number SG0025). Perform PCR using ExTaq DNA polymerase (Takara Shuzo, Catalog No. RR001A) from 834 bp upstream to 34 bp downstream from the transcription start point using the collected DNA of 2 as a template (Figure 5 shows the primer sequence and PCR conditions) , S-OCp, SEQ ID NO: 15, AS-OCp, SEQ ID NO: 16), an 868 bp PCR product was obtained. This was subjected to electrophoresis and purification, and then directly inserted into the T vector pGEM-T Easy (Promega, catalog number A1360) by ligation with T4 DNA ligase as the OC promoter. After confirming that the cloned OC promoter had the correct nucleotide sequence with a DNA sequencer, it was cut out from the vector by NotI digestion and blunt-ended with T4 DNA polymerase. pΔPr.-E1A-CMV-19K and pΔPr.-E1AΔ24-CMV-19K were digested with SalI, blunt-ended with T4 DNA polymerase, treated with CIP, and inserted with the above-mentioned OC promoter by ligation reaction with T4 DNA ligase. POC-E1A-CMV-19K and pOC-E1AΔ24-CMV-19K were prepared. The pOC-E1A-CMV-19K and pOC-E1AΔ24-CMV-19K prepared in this way are growth control vector plasmids in which E1A or E1AΔ24 is expressed by the OC promoter and E1B19K is expressed by the CMV promoter ( (See Figures 6-1 and 6-2).

Example 3 (Preparation of a vector plasmid containing a therapeutic gene expression unit)
A plasmid that can be freely inserted into the growth control type vector plasmid in advance or into the growth control type adenovirus vector plasmid in which recombination has been completed was prepared using the Cre recombination reaction. A plasmid having a LoxP sequence and incorporating a therapeutic gene and its promoter was prepared as follows. The Kn (kanamycin) resistance gene of pUni / V5-HisC (Invitrogen, Carlsbad, CA, product number ET003-11) was excised with BglII (Toyobo, catalog number BGL-211) and SmaI (Toyobo, catalog number SMA-111). It was blunt-ended with T4 DNA polymerase and treated with CIP to prevent self-ligation. After digesting the Tc (tetracycline) resistance gene from pBR322 (Toyobo, catalog number DNA-003) with SspI (Toyobo, catalog number SSP-101) and StyI (New England Biolabs, Beverly, MA, catalog number R0050S), T4 Blunt ends were made with DNA polymerase. This gene is inserted into pUni / V5-HisC, which has been blunt-ended with the Kn resistance gene removed as described above, by ligation using T4 DNA ligase, and the Kn resistance gene is replaced with the Tc resistance gene. A vector plasmid pUni / V5-HisC-Tc containing an expression unit was prepared (see FIG. 7-1). Further, this pUni / V5-HisC-Tc was digested with XhoI, blunt-ended with T4 DNA polymerase, and treated with CIP. On the other hand, as described above, using the plasmid pRc / CMV as a template, a sense primer (S-CMVp, SEQ ID NO: 11) with a SalI site and an antisense primer (AS-CMVp, SEQ ID NO: 12) with a MfeI site. The CMV promoter part obtained by PCR in the above was excised with SalI and MfeI enzymes, blunt-ended with T4 DNA polymerase, and the above blunt-ended pUni / V5-HisC-Tc with T4 DNA ligase. Ligation reaction was performed to prepare pUni / V5-HisC-Tc-CMV. In other words, pUni / V5-HisC-Tc-CMV is a plasmid in which the CMV promoter is inserted into pUni / V5-HisC-Tc, and the target therapeutic gene to be expressed downstream of the CMV promoter is AgeI of the multicloning site, Insertion can be performed using either ApaI or StuI. The CMV promoter and the gene inserted downstream thereof are inserted into the growth control type vector plasmid controlling the E1 part described in the previous section by Cre expression, or finally the growth control type adenovirus vector plasmid completed. In addition, a therapeutic gene can be inserted by Cre expression. In addition, the CMV promoter can be easily excised with the restriction enzyme HincII-AgeI when necessary, and a tissue-specific promoter can be easily inserted into that portion (see FIG. 7-1).

Example 4 (Preparation of first therapeutic gene expression vector plasmid)
As an example of this, a vector plasmid into which EGFP (enhanced green fluorescent protein) serving as a marker gene was inserted was prepared as follows. First, pEGFP-C1 (CLONETECH, catalog number 6084-1) was digested with BclI, blunt-ended with T4 DNA polymerase, and then EGFP cDNA was excised by AgeI digestion. On the other hand, pUni / V5-HisC-Tc-CMV was digested with ApaI, blunt-ended with T4 DNA polymerase, digested with AgeI, and the excised EGFP cDNA fragment was ligated with T4 DNA ligase. To obtain pUni / V5-HisC-Tc-CMV-EGFP. In other words, the first therapeutic gene expression vector plasmid pUni / V5-HisC-Tc-CMV-EGFP is an expression vector that can express EGFP from the CMV promoter, and a growth control vector plasmid that controls the E1 part, and finally The CMV-EGFP gene can be inserted into the completed growth-regulated adenovirus vector plasmid by Cre recombination reaction (see Fig. 7-1). EGFP is not a therapeutic gene, but is inserted as an alternative to the therapeutic gene for experimental convenience.

Example 5 (Recombination by recombination of growth control vector plasmid and first therapeutic gene expression vector plasmid, preparation of second therapeutic gene expression vector plasmid)
A growth control vector plasmid (expressing E1A and E1B with various promoters) and a first therapeutic gene expression vector plasmid (expressing a therapeutic gene with various promoters) can be easily recombined with Cre recombinase (LoxP , The reaction of recognizing the LoxH sequence and linking two plasmids into one). In this example, the following 8 combinations were recombined. Growth control vector plasmids (pCEA-E1A-CMV-19K, pCEA-E1AΔ24-CMV-19K, pE2F-E1A-CMV-19K, pE2F-E1AΔ24-CMV-19K, pCEA-E1A-E2F-19K, pCEA-E1AΔ24- 100 ng each of E2F-19K, pOC-E1A-CMV-19K, pOC-E1AΔ24-CMV-19K) and the first therapeutic gene expression vector plasmid (pUni / V5-HisC-Tc-CMV-EGFP), Cre recombinase ( React with Invitrogen, catalog number R100-10) (37 ° C, 20 minutes). Next, the reaction was stopped by inactivating Cre by treating at 65 ° C. for 5 minutes. This reaction solution was transformed into competent cells DH5α (Toyobo, catalog number DNA-903) and LB (Luria-Bertani, nacalai tesque, containing tetracycline (7.5 μg / ml, Wako Pure Chemicals, catalog number 205-08591), Catalog number 20066-95) Incubate on agarose plate. The growth control vector plasmid cannot form a colony because it is a Kn resistance gene, and the first therapeutic gene expression vector plasmid also has a special Ori (E. coli replication origin) R6Kγ, so it cannot form a colony with DH5α. In contrast, since the second therapeutic gene expression vector plasmid proliferation-regulated vector plasmid and a first therapeutic gene expression vector plasmid was prepared by Recombination is with both pUC Ori and Tc ^r, forming colonies (See Fig. 7-2. In the figure, the second treatment is made by recombination of pPrA-E1A-PrB-E1B and pUni / V5-HisC-Tc-CMV-GENE.) Gene expression vector plasmid). Initially, the above reaction was performed using Cre recombinase (Invitrogen, catalog number R100-10) purchased from Invitrogen, but colonies of E. coli were not obtained on the LB plate. Using the plasmid pUni / V5-HisC and pcDNA4 / HisMax-E included in the Invitogen recombination kit (Echo cloning system, catalog number ET401-30C) to confirm that the reaction system is working correctly As a result of the above reaction, only 5 or less colonies were obtained. That is, the activity of Cre recombinase was considered to be low. Therefore, Cre recombinase was extracted using an adenoviral vector AxCANCre (provided by RIKEN DNA Bank) that expresses the Cre gene. First, HepG2 cells, which are human hepatoma cells with high gene transfer efficiency by adenovirus (provided by Medical Cell Resource Center, Institute of Aging Medicine, Tohoku University) 30 MOI in one 10 cm diameter culture dish
(Multiple infection degree, 1 MOI = 1 plaque forming unit / cell), 3 days later, the cells were detached by trypsin (nacalai tesque, catalog number 35555-54) treatment, washed with PBS, and then lysed buffer (20 mM Tris It was dissolved in 200 μl of pH 7.5, 150 mM NaCl, 10 mM MgCl ₂ , 10% glycerol, 1% protease inhibitor cocktail (SIGMA, catalog number P2714)), and the one obtained by repeating freezing and thawing three times was used as Cre recombinase. When this Cre was used for the reaction, colonies were obtained, and all the appearing colonies were positive clones. In other words, Cre extracted using AxCANCre was shown to have sufficient activity in this reaction system. The second therapeutic gene expression vector plasmids obtained by this reaction are pCEA-E1A-CMV-19K / CMV-EGFP, pCEA-E1AΔ24-CMV-19K / CMV-EGFP, pE2F-E1A-CMV-19K / CMV, respectively. -EGFP, pE2F-E1AΔ24-CMV-19K / CMV-EGFP, pCEA-E1A-E2F-19K / CMV-EGFP, pCEA-E1AΔ24-E2F-19K / CMV-EGFP, pOC-E1A-CMV-19K / CMV-EGFP POC-E1AΔ24-CMV-19K / CMV-EGFP.

Example 6 (Recombination into Adenovirus Genome, Production of Growth Control Type Adenovirus Vector Plasmid)
In parallel with the second therapeutic gene expression vector plasmid (the above-mentioned Cre recombination), a growth control-type adenovirus vector plasmid having no therapeutic gene was prepared to analyze the system and functions. The 30.3 kb plasmid pAdHM4 (provided by Dr. Mark A. Kay, Stanford University) carrying the adenovirus genome (E1 region 342-3523 bp, part of E3 region 28133-30818 bp deleted) was deleted E1 It has restriction enzyme I-CeuI and PI-SceI recognition sequences for inserting foreign genes into this part. First, pAdHM4 was digested with restriction enzyme I-CeuI (New England Biolabs, catalog number R0699S) (0.2 U / μg DNA, 37 ° C., 1 hour), phenol / chloroform purified, and ethanol precipitated. Next, it was digested with PI-SceI (New England Biolabs, catalog number R0696S) and purified in the same manner. pCEA-E1A-CMV-19K, pCEA-E1AΔ24-CMV-19K, pE2F-E1A-CMV-19K, pE2F-E1AΔ24-CMV-19K, pCEA-E1A-E2F-19K, pCEA-E1AΔ24-E2F-19K, pOC- E1A-CMV-19K and pOC-E1AΔ24-CMV-19K were also digested with I-CeuI and PI-SceI, respectively, and inserted into pAdHM4 using T4 DNA ligase, respectively. The obtained plasmid was always digested with I-CeuI and PI-SceI to confirm the inserted gene. The thus obtained growth control type adenovirus vector plasmids were pAd.HM4-CEA-E1A-CMV-19K, pAd.HM4-CEA-E1AΔ24-CMV-19K, pAd.HM4-E2F-E1A-CMV-19K, pAd. HM4-E2F-E1AΔ24-CMV-19K, pAd.HM4-CEA-E1A-E2F-19K, pAd.HM4-CEA-E1AΔ24-E2F-19K, pAd.HM4-OC-E1A-CMV-19K, pAd.HM4- OC-E1AΔ24-CMV-19K. These plasmids have a portion other than E1 and E3 of the adenovirus genome and a portion that expresses the E1A and E1B19K regions with a foreign promoter.

Example 7 (transfection into 293 cells)
The growth-regulated adenovirus vector plasmid obtained in Example 6 was transfected into 293 cells derived from human fetal kidney cells. As a culture solution of 293 cells, DMEM (Dulbecco's Minimum Essential Medium, SIGMA, catalog number D5796) containing inactivated 10% fetal calf serum (MBL, catalog number 268-1) was used. Transfection is based on the calcium phosphate method (Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 9.1.4-9.1.9, 1999). The adenovirus genome is contained in 1.5 ml of HEPES buffered saline (pH 7.1). Add 40 μg of total DNA of 20 μg of linear DNA and 20 μg of testicular DNA (SIGMA, catalog number D-7656), add 75 μl of 2.5 M CaCl ₂ while stirring, and let stand at room temperature for 30 minutes. The culture solution was added dropwise with stirring. After 4 hours and a half in a CO ₂ incubator, α-MEM (GIBCO BRL, catalog number 12000-022) plus 10% inactivated horse serum (GIBCO BRL, catalog number 26050-088) plus 1% A solution in which equal amounts of agarose gel were mixed was layered on a culture dish, and 293 cells were continuously cultured on a solid medium. In order to evaluate the introduction efficiency, pEGFP-C1 was transfected with the same protocol, and after 48 hours, positive cells were counted under a fluorescence microscope. As a result, gene was introduced into about 30% of cells each time. When adenovirus is produced, 293 cells are cytopathic (CPE:
A plaque is formed by a cytopathic effect.
In the conventional method in which adenovirus is produced by homologous recombination, transfection in a large number of culture dishes and high gene transfer efficiency are required due to the low efficiency of homologous recombination. In vitro ligation method In the production method of the present invention based on (Mizuguchi, H., et al, Hum. Gene Ther., 10, 2013-2017, 1999), more than 30 plaques appeared in a 10 cm culture dish, with an introduction efficiency of 30%. It is enough.

Example 8 (Two variants for rapid recombination of a growth-regulated adenovirus vector plasmid incorporating a therapeutic gene)
1. System for recombination of therapeutic genes by direct recombination of growth control adenovirus vector plasmid and first therapeutic gene expression vector plasmid Growth control vector plasmid (eg pCEA-E1A-CMV-19K) that does not have a therapeutic gene If only the therapeutic gene can be freely incorporated after recombination into an adenovirus vector plasmid (such as pAdHM4), recombination is faster and various combinations of ADVs can be produced easily. In order to establish this system, recombination was first performed using pAd.HM4-CEA-E1A-CMV-19K and pUni / V5-HisC-Tc-CMV-EGFP as an example. 100 ng of each of these two plasmids is mixed and reacted (37 ° C., 20 minutes) using Cre recombinase purified as described above. Subsequently, the reaction was terminated by inactivating Cre by treating at 65 ° C. for 5 minutes. This reaction solution was transformed into competent cells DH5α (Toyobo, catalog number DNA-903) and LB (Luria-Bertani, nacalai tesque, containing tetracycline (7.5 μg / ml, Wako Pure Chemicals, catalog number 205-08591), Catalog number 20066-95) Incubate on agarose plate. The next day, 5 colonies appeared on the plate. When the plasmid obtained from this colony was analyzed, the growth control-type adenovirus vector plasmid pAd.HM4-CEA-E1A-CMV-19K / in which the therapeutic gene into which the CMV-EGFP gene had been inserted was incorporated. CMV-EGFP. The reason for the small number of colonies is that the adenoviral vector plasmid is as long as 30 kb and the effect of the antibiotic tetracycline, but the probability of inserting a therapeutic gene from this result is 100%. It is only necessary to obtain one colony.

  With this modification, when E1A and E1B promoters are fixed and various therapeutic genes and their promoters are compared to compare the effects, it is no longer necessary to insert the therapeutic gene into the shuttle vector and then recombine with the adenoviral vector each time. .

2. Intracellular Cre recombination by co-transfection Express the first therapeutic gene expression vector plasmid without recombination into the growth control adenovirus vector plasmid pAd.HM4-CEA-E1A-CMV-19K By co-transfecting the cells, the two plasmids were recombined in the cells, and an adenovirus with a therapeutic gene was obtained. Calcium phosphate method using 20 μg each of pAd.HM4-CEA-E1A-CMV-19K and pUni / V5-HisC-Tc-CMV-EGFP expressing EGFP gene as a marker in 293 cells that constantly express Cre Co-transfected with In order to evaluate gene transfer efficiency, the plasmid pEGFP-C1 expressing the EGFP gene was transfected by the same method, the medium was replaced with a new one after 4 and a half hours, and the cells were collected with trypsin after 48 hours, and fluorescence microscopy When EGFP positive cells were counted below, the positive rate (= gene transfer efficiency) was 21%. Plaques appeared after 6 days, 10 complete plaques after 12 days and 10 plaques after 14 days. Among these plaques, those with ADV in which Cre recombination has occurred and EGFP has been incorporated are observed to be positive for EGFP when observed under a fluorescence microscope. This time, only one of the 10 plaques was positive for EGFP, so the probability of ADV incorporating EGFP appears to be 10%. In this experimental system, the number of plaque appearances is smaller than that in the normal ADV preparation method described above, because when the plasmid containing ADV genomic DNA is linearized by digestion with the restriction enzyme PacI, Cre recombination does not occur. It is thought that the reason is that the efficiency of ADV generation is reduced by removing PacI digestion. Of the EGFP (+) plaque collection solution and the EGFP (-) plaque collection solution, a total of 4 clones, 3 clones, were infected and amplified in 24 wells of 293 cells. In the EGFP (+) clone, cells in all wells became EGFP positive, whereas in other wells, only some cells were EGFP positive. CPE appeared in all clones after 3 days and was recovered. As a next step, 293 cells in a 10cm dish were infected. Even in the EGFP (+) clone, only about 50 to 60% of the cells were positive for EGFP in the first day after infection, and on the second day all cells were CPE. However, only 80% of the cells were EGFP positive. This means that the remaining 20% of the cells are CPE due to ADV that does not have the EGFP gene, and it is possible that ADV of EGFP (-) was mixed in plaques collected as monoclonals. It was. In order to purify this ADV solution, it is considered that plaques that become EGFP positive by infecting 293 cells again may be collected.

  From the above, it was confirmed that by this cotransfection method, ADV having an inserted gene (EGFP gene) can be obtained by recombining two plasmids in Cre-expressing 293 cells. This method is simpler and quicker than a system in which Cre reaction is performed in a test tube, E. coli is transformed to obtain a plasmid, and then transfected.

Example 9 (Collection, propagation and purification of growth-regulated recombinant adenovirus vector from plaque)
12 Plaques appeared 7 days after transfection with Ad.HM4-CEA-E1A-CMV-19K. Collect the plaque as a solid medium using a 1,000 μl micropipette tip, with a thickened tip, and `` complete plaque formation '' when the cells disappear completely at the center of the plaque and the bottom of the culture dish is visible. And suspended in 800 μl medium and stored at −80 ° C. Ten plaques were collected for each virus.

Next, 600 μl of the adenovirus solution obtained by disrupting the cells by repeating the cycle of thawing this solution in a 37 ° C. constant temperature bath and freezing with liquid nitrogen three times was seeded at 1 × 10 ⁵ in a 24-well culture plate the day before. Infect infected 293 cells. After infection, 200 μl of medium is added every 3 days, and CPE appears in 5-7 days, although there are variations. After the appearance of CPE, collect both medium and cells and store at -80 ° C. Next, 800 μl of this solution (80% of the total volume) is similarly frozen and thawed three times, and then infected with 70 to 80% confluent 293 cells in a 10 cm culture dish. At this stage, CPE appears in 2-4 days, and the medium and cells are collected and stored at -80 ° C. The next step is to freeze and thaw this solution three times, and then infect 80% (usually 8 ml) of the total volume to 70-80% confluent 293 cells in 15 cm culture dishes (six). CPE appears 2-3 days after infection and media and cells are collected and stored at -80 ° C. Finally, after freezing and thawing this solution three times, 80% of the total volume (usually 100 ml) is infected with 40 to 15 cm culture dishes with -80 80% confluent 293 cells. Since CPE usually appears in 2 days here, the medium and cells are collected, centrifuged at 1000 rpm, the supernatant is removed, and 1.5 ml of PBS (phosphate buffered saline) per 50 ml of cell suspension. ), And store 40 cells as a 24 ml cell suspension at -80 ° C. (At this time, if one 45 ml of the supernatant is stored without discarding all the supernatant, amplification is possible by infecting 40 pieces of 15 cm culture dishes using this solution.) Cell floating The solution is frozen and thawed three times, and then centrifuged at 1,000 rpm to collect the supernatant. Density of cesium chloride (Wako Pure Chemicals, catalog number 039-01955) in a Hitachi ultracentrifuge tube (Hitachi Koki, catalog number S303276A) Prepare 4 gradient solutions (1.5g / ml CsCl 0.5ml, 1.35g / ml CsCl 3ml, 1.25g / ml CsCl 3ml) and put 6ml of virus solution in each. After strict balance with PBS, first centrifuge at 10 ° C, 35,000 rpm for 1 hour using a swing rotor (Hitachi Koki, RPS40T) in a cooling high-speed centrifuge (Hitachi Koki, himac CP65β). Three bands are usually visible, but the pale blue band at the bottom is adenovirus. Carefully remove the supernatant before collecting this layer in a volume of 1 ml (up to 1.5 ml). A total of about 4 ml (up to 6 ml) of virus solution can be obtained. Next, put this virus solution into a centrifuge tube containing 1.35 g / ml CsCl 6 ml. Similarly, balance the balance tube with 1.35 g / ml CsCl in the same way, at 10 ° C, 35,000 rpm, over 18 hours. Centrifuge. The virus now appears as a single band, and after removing the supernatant, collect the virus solution in a volume of 1-2 ml.

Then, it refine | purified in the desalting column as follows. Discard the contents of the desalting column (Biorad, Econopac 10DG, product number 732-2011), add 15 ml of PBS, and add all of the solution dropwise. Add Xml of virus solution here. Collect 1 ml of the eluate from the column in a 1.5 ml microtube with a number (1-7). Next, add (3-X) ml of PBS to the column and collect 1 ml in the same manner. Add 4 ml of PBS and collect 1 ml in the same manner. This gives a total of 7 from 1 to 7, but the 4th tube contains most of the concentrated virus. As a precaution, measure the ADV concentration in all tubes by the following method. Mix 94 μl of PBS, 5 μl of 10% SDS and 1 μl of virus solution, and mix well by vortexing (SCIENTIFIC INDUSTRIES, Inc., BOHEMIA, NY) to destroy the outer shell of the virus. This is centrifuged at 15,000 rpm for 3 min, the supernatant is collected, and OD _{260 nm} is measured. Calculate the concentration of the virus solution as 1 OD _260nm = 1 x 10 ¹² particles / ml (1 OD _260nm = 2 x 10 ¹³ particles / ml for a 20-fold dilution). Usually, when amplified on 40 15 cm culture dishes, 1.5-2 ml of virus solution was obtained at a concentration of 1-5 × 10 ¹² particles / ml.

  The purified ADV solution was dispensed with 10% Glycerol (Wako Pure Chemicals, catalog number 075-00616), and stored at −80 ° C. Thus, in Ad.CEA-E1A-CMV-19K and similar methods, Ad.CEA-E1AΔ24-CMV-19K, Ad.E2F-E1A-CMV-19K, Ad.E2F-E1AΔ24-CMV-19K, Ad.CEA- Eight types of adenoviruses were obtained: E1A-E2F-19K, Ad.CEA-E1AΔ24-E2F-19K, Ad.OC-E1A-CMV-19K, Ad.OC-E1AΔ24-CMV-19K.

Example 10 (Proliferation Confirmation of Produced Growth Controlled Recombinant Adenovirus Vector)
As an example, an experiment was conducted to confirm the CEA promoter-dependent growth of Ad.CEA-E1A-CMV-19K. CEA-expressing colorectal cancer cell lines colo-205, Lovo, HCT-15 cells (provided by the Medical Cell Resource Center, Institute of Aging Medicine, Tohoku University), non-CEA-expressing Hela cells, and all adenoviruses grow as positive controls Each of 293 cells was infected with Ad.CEA-E1A-CMV-19K at MOI 100, 10, 1, 0.1, 0.01, 0, and the appearance of CPE was observed. On the second day after infection, all cells showed strong cytotoxicity in the wells with MOI 100. In contrast, wells infected with non-proliferating (E1-deficient) ADV, Ad.CMV-LacZ, showed mild cytotoxicity even at MOI 100, and CEA-expressing colon cancer cell lines already had viral growth. It was thought to have started. 7 days after infection, CPE up to MOI 1 in Lovo and 293, CPE up to MOI 10 in HCT-15, mild cytotoxicity at MOI 100 in colo-205, CPE only in MOI 100 in Hela, Lovo has the same CPE as 293 ADV proliferated to some extent, and HCT-15 also proliferated to the next level, and it was considered that there was no obvious ADV proliferation in colo-205 and Hela. Eventually, two weeks after infection, cell damage was observed in Lovo up to MOI 0.1, HCT-15 up to MOI 1, and 293 up to MOI 0.01, indicating the degree of ADV proliferation. On the other hand, no cell damage was observed with MOI 100 in colo-205, but the cell damage was observed up to MOI 10 in Hela, indicating that the growth of ADV was restricted by the CEA promoter. it is conceivable that. However, it is due to the growth of ADV that some cell damage was seen in Hela of CEA non-producing cancer,
To determine whether it is due to Hela sensitivity, it is necessary to be able to evaluate the growth of ADV over time. Therefore, an EGFP gene as a marker was incorporated into a vector into which a therapeutic gene was incorporated to produce Ad.CEA-E1A-CMV-19K / CMV-EGFP. This ADV can know the growth of ADV by observing the positive rate of EGFP after infecting cells. Table 1 below is a graph showing the cell proliferation (CPE) proliferation ability 12 days after the infection with the growth control recombinant adenovirus vector. “Intact” means invariant (no injury), and the same applies to Tables 2 and 3 below.

Example 11 (Proliferation-controlled recombinant adenovirus vector expressing EGFP)
Ad.CEA-E1A-CMV-19K / CMV-EGFP was prepared as described above and purified by ultracentrifugation.
2 ml of virus solution was obtained at a concentration of 10 ¹² particles / ml. CEA-expressing cancer cells MKN-1, MKN-28, MKN-45, HCT-15, Lovo, colo-205 were infected with MOI 100, 10, 1, 0.1, 0.01, 0 and observed for EGFP positive rate under a fluorescence microscope However, ADV proliferation was assessed by the appearance of cytotoxicity (CPE). Two days after infection, except for colo-205, almost 100% of cells were EGFP positive with MOI 100, and only about 50% of colo-205 cells were EGFP positive. That is, colo-205 means that the gene transfer efficiency by ADV is low. Over time, both EGFP positive rate and CPE increased, but the trend was strong for MKN-28, Lovo, and HCT-15. As an example, the result of MKN-28 shows that about 20% of the well of MOI 1 was EGFP positive after 1 day of infection and no cell damage was seen, but the EGFP positive rate was about 50% after 6 days. After 8 days, cell damage (some cells became CPE) appeared, and after 12 days, more than 80% of the cells became positive, and almost became CPE. In other words, the growth of ADV observed with the marker gene EGFP and CPE, which indicates cell damage, appeared in correlation, so Ad.CEA-E1A-CMV-19K / CMV-EGFP grew in CEA-producing cancer. It was proved that its toxicity can kill cells. Tables 2 and 3 below are graphs showing the proliferation ability of the growth-regulated recombinant adenovirus vector 12 days after infection as a positive rate of EGFP and the proliferation ability 12 days after infection as a cell death (CPE), respectively. is there.

It is the schematic which shows the principle of the efficient preparation method of the growth control type | mold recombinant adenovirus vector of this invention. The PCR primer used for preparation of the vector plasmid containing the unit for growth control of Example 1 and the conditions of PCR reaction are shown. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an explanatory diagram illustrating the principle of a mutation introduction method for preparing a vector plasmid containing a growth control unit lacking the Rb protein binding sequence of Example 1. The PCR primers and PCR reaction conditions used in the preparation of the vector plasmid containing the growth control unit lacking the Rb protein binding sequence of Example 1 and the vector plasmid containing the growth control unit having the entire E1B region are shown. The PCR primers and PCR reaction conditions used for the production of the growth control vector plasmid of Example 2 are shown. The growth control type | mold vector plasmid which does not delete the Rb protein binding site produced in Example 2 is shown. The growth control type | mold vector plasmid which deleted the Rb protein binding site produced in Example 2 is shown. The vector plasmid containing the therapeutic gene expression unit produced in Example 3 and the first therapeutic gene expression vector plasmid produced in Example 4 are shown. The process by which the 2nd therapeutic gene expression vector plasmid of Example 5 is produced by recombination is shown.

Claims (15)

  In order from the upstream, the E1A region, at least one protein coding region of the E1B region or the entire E1B region, a poly A signal sequence, and a recombinase recognition sequence, the endogenous promoter of the E1A region and at least one of the E1B regions A vector plasmid comprising a growth control unit prepared by deleting an endogenous promoter that regulates the expression of a gene encoding a protein in the protein coding region and inserting a restriction enzyme recognition sequence at each of these deletions. Adenovirus prepared by introducing a promoter specifically expressing in the target tissue into the restriction enzyme recognition sequence to prepare a growth control vector plasmid, and further deleting the growth control vector plasmid from the E1 region. Integrated into a vector plasmid having a genome, Efficient manufacturing method of growth control recombinant adenovirus vector characterized by producing fertilized control adenoviral vector plasmids.   The method for efficiently producing a growth-regulated recombinant adenovirus vector according to claim 1, wherein the E1A region lacks the Rb protein binding sequence.   The method for efficiently producing a growth-regulated recombinant adenovirus vector according to claim 1 or 2, wherein the protein coding region of the E1B region is a 19 KDa protein coding region and / or a 55 KDa protein coding region.   A restriction enzyme that is blunt ended in a restriction enzyme recognition sequence that is inserted into a deletion site of the endogenous promoter that regulates the expression of a gene encoding a protein in the endogenous promoter of the E1A region and at least one protein coding region of the E1B region The method for efficiently producing a growth-regulated recombinant adenovirus vector according to any one of claims 1 to 3, wherein each site is contained. Efficient method for manufacturing a proliferation-regulated recombinant adenoviral vectors according recombinase recognition sequence on either of the claims 1 to 4 is a variant sequence of LoxP addition Waso.   The restriction of the vector plasmid comprising the growth control-type vector plasmid according to any one of claims 1 to 5 and a therapeutic gene expression unit prepared by inserting a recombinase recognition sequence and a restriction enzyme recognition sequence in order from upstream. Recombinase is allowed to act on a first therapeutic gene expression vector plasmid prepared by introducing either a constitutive strong expression promoter or a therapeutic gene expression promoter and a therapeutic gene in order from the upstream into the enzyme recognition sequence, thereby allowing the second treatment. A gene expression vector plasmid is prepared, and further, this second therapeutic gene expression vector plasmid is incorporated into a vector plasmid having an adenovirus genome prepared by deleting the E1 region, and a growth control type adenovirus into which the therapeutic gene is incorporated Produced by vector plasmid Efficient method for manufacturing a proliferation-regulated recombinant adenoviral vectors therapeutic gene is incorporated to.   A vector plasmid comprising a growth control type adenovirus vector plasmid according to any one of claims 1 to 5 and a therapeutic gene expression unit prepared by inserting a recombinase recognition sequence and a restriction enzyme recognition sequence in order from the upstream. A recombinase is allowed to act on the restriction enzyme recognition sequence to a first therapeutic gene expression vector plasmid prepared by introducing either a constitutive strong expression promoter or a therapeutic gene expression promoter and a therapeutic gene in order from upstream. An efficient method for producing a growth-regulated recombinant adenovirus vector incorporating a therapeutic gene, comprising producing a growth-regulated adenovirus vector plasmid incorporating the gene.   The growth incorporating a therapeutic gene according to claim 7, wherein the growth control-type adenovirus vector plasmid and the first therapeutic gene expression vector plasmid are mixed to allow recombinase to act, and then both are transformed. An efficient method for producing a controlled recombinant adenovirus vector.   8. The growth-regulated recombinant adeno incorporating the therapeutic gene according to claim 7, wherein the growth-regulated adenovirus vector plasmid and the first therapeutic gene expression vector plasmid are cotransfected into a cell expressing recombinase. An efficient method for producing a viral vector.   10. A method for efficiently producing a growth-regulated recombinant adenovirus vector incorporating a therapeutic gene according to claim 9, wherein the recombinase-expressing cell is obtained by expressing the recombinase in a cell expressing the adenovirus E1 region protein. .   The therapeutic gene according to any one of claims 6 to 10, wherein the recombinase recognition sequence of the vector plasmid containing the therapeutic gene expression unit is other than the recombinase recognition sequence of the vector plasmid containing the growth control unit. An efficient method for producing a growth-regulated recombinant adenovirus vector. Different drug resistance gene of the vector plasmid containing the drug resistance gene of the vector plasmid having a proliferation-regulating unit and the therapeutic gene expression unit and, Ori vector plasmid containing a therapeutic gene-expressing unit expresses p ir gene competent The method for efficiently producing a growth-regulated recombinant adenovirus vector into which a therapeutic gene according to any one of claims 6 to 11 is incorporated, wherein the method can be replicated only in tent cells.   A vector plasmid comprising a growth control unit for use in an efficient production method of the growth control type recombinant adenovirus vector according to any one of claims 1 to 5.   A vector plasmid containing a growth control unit and an adenovirus genome lacking the E1 region for use in the efficient production method of the growth control type recombinant adenovirus vector according to any one of claims 1 to 5. And a vector kit.   A vector plasmid comprising a growth control unit for use in an efficient production method of a growth control type recombinant adenovirus vector into which the therapeutic gene according to any one of claims 6 to 12 is incorporated, and a therapeutic gene expression A production kit comprising a vector plasmid comprising a unit and a vector plasmid having an adenovirus genome lacking at least the E1A region.

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